title: Bacterial expression and purification of active hematopoietic cell kinase.
abstract: Src family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs. So far, high-yield bacterial expression systems have only been achieved for SFK kinase domains or for inactive mutants of constructs containing the regulatory SH3 and SH2 domains, but not for their active forms. Herein described is a bacterial expression system for the wild type, active SFK Hck containing SH3, SH2 and kinase domains. Hck plays an important role in phagocyte function as well as the etiology of chronic myeloid leukemia as Hck is an interaction partner of Bcr-Abl. Structural studies of Hck are essential to fully understand the signaling processes involved in host defense and leukemogenesis. Successful bacterial expression of Hck was possible by a dual strategy: (1) co-expression with YopH phosphatase in order to control host toxicity, and (2) expression in a bacterial strain that is RNase E deficient, which dramatically increased overall expression levels. The expressed Hck construct is unphosphorylated and appears to be in an open conformation. Bacterially expressed Hck is capable of autophosphorylation, phosphorylates substrate at rates comparable to insect cell expressed Hck, and can be inhibited by staurosporine and Csk.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia.
authors.forename: Romana
authors.initials: R
authors.lastname: Kristelly
authors.forename: Theresa Wenli
authors.initials: TW
authors.lastname: Qiu
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Gunn
authors.forename: Denis B
authors.initials: DB
authors.lastname: Scanlon
authors.forename: Terrence D
authors.initials: TD
authors.lastname: Mulhern
mesh_terms: D000818:Animals; D001709:Biotechnology; D015153:Blotting, Western; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051572:Proto-Oncogene Proteins c-hck / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018909:src Homology Domains
chemical_list: D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck